A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

Background: The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood. Methods: We constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G(0)-arrested cells. Results: MCMV replicated to higher titers in G(0)-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G(0)-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G(0) by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells. Conclusion: By means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells

Murine cytomegalovirus infection via the intranasal route offers a robust model of immunity upon mucosal CMV infection.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. The experimental study of immunity against CMV in animal models of infection, such as the infection of mice with the mouse CMV (MCMV), has relied on systemic intraperitoneal infection protocols, although the infection naturally transmits by mucosal routes via body fluids containing CMV. To characterize the biology of infections by mucosal routes, we have compared the kinetics of virus replication, the latent viral load, and CD8 T cell responses in lymphoid organs upon experimental intranasal and intragastric infection to intraperitoneal infection of two unrelated mouse strains. We have observed that intranasal infection induces robust and persistent virus replication in lungs and salivary glands, but a poor one in the spleen. CD8 T cell responses were somewhat weaker than upon intraperitoneal infection, but showed similar kinetic profiles and phenotypes of antigen-specific cells. On the other hand, intragastric infection resulted in abortive or poor virus replication in all tested organs, and poor T cell responses to the virus, especially at late times after infection. Consistent with the T cell kinetics, the MCMV latent load was high in the lungs, but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we show here that intranasal, but not intragastric infection of mice with MCMV represents a robust model to study short and long-term biology of CMV infection by a mucosal route

IFNβ suppresses MCMV IE gene expression at the transcriptional level by inducing ND10 genes.

<p>LSECs were treated with medium ± IFNβ (500 U/mL) and 24 h later infected with MCMV WT. Virus absorption was restricted to 5 minutes to improve time resolution. Nascent RNA samples were collected at 1 hpi and used for deep-sequencing. (<b>A</b>) Histograms show normalized reads of viral transcripts in IFNβ-treated and untreated LSECs from two replicates. The fraction of the viral transcriptome corresponding to ie1, ie2 or ie3 transcripts is indicated (<b>B</b>) Counts of normalized Daxx, Sp100 and PML transcripts in IFNβ-treated and non-treated LSECs. Histograms show normalized reads from two replicates (<b>C</b>) LSECs were treated with 10, 100 or 500 U/mL IFNβ or left untreated for 24 h and then stained for Daxx. Representative fluorescent pictures are shown. (<b>D</b>) LSECs were transfected with plasmids expressing shRNA against Daxx, Sp100 or PML, treated with IFNβ or left untreated and infected 24 h later with 0.01 MOI of MCMV^r. Control cells were transfected with scrambled shRNA in the presence or absence of IFNβ and infected as above. Viral plaques were counted 4 dpi, normalized to represent IFNβ-untreated samples as 100 and average normalized PFU from three independent experiments ± SD are shown. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003962#ppat.1003962.s003" target="_blank">Figure S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003962#ppat.1003962.s006" target="_blank">Table S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003962#ppat.1003962.s007" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003962#ppat.1003962.s008" target="_blank">S3</a>.</p

IFNβ suppresses MCMV immediate early gene expression and not virus entry.

<p>(<b>A</b>) MEF expressing Cre recombinase were treated with IFNβ and infected with WT or IE1/3^flox MCMV (0.0001 MOI). At 7 dpi, IFNβ was removed in selected wells (± IFNβ) or resupplied at regular intervals until day 28 (+IFNβ). Control cells were infected in the absence of IFNβ (−IFNβ). The mean percentage of wells showing viral plaques ± SEM at indicated time points from three independent experiments is shown. (<b>B</b>) MEFs were transfected with the MCMV^r BAC and supplied with IFNγ 5 h later. After 4 days, the wells were inspected for signs of EYFP expression and wells were either resupplied with IFNβ (+IFNβ) or, in selected wells, it was removed from the cell medium (± IFNβ). Wells were reassessed for EYFP-expression at 6 days post transfection. The percentage of positive wells showing EYFP-expression from two independent experiments (± SD) is shown. (<b>C</b>) NIH3T3 fibroblasts were transfected with a plasmid expressing EYFP and tdTomato driven by the MCMV MIEP promoter. The cells were treated with IFNβ at 5 h post transfection and analyzed by fluorescence microscopy and flow cytometry at 3 days post transfection. Representative fluorescent images are shown and histograms indicate the percentage of transfected cells (EYFP+tdTomato) from triplicates (± SD). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003962#ppat.1003962.s002" target="_blank">Figure S2</a>.</p

IFNβ restricts MCMV replication in LSECs.

<p>LSECs were incubated for 24(+IFNβ, 500 U/mL) or without IFNβ (−IFNβ), and infected with MCMV. The same medium was added following infection. (<b>A</b>) Supernatants (SN) from cells infected at an MOI 0.001 were collected daily up to 7 dpi and titrated on IFNAR^−/− MEFs. The average titers (PFU/mL) from triplicates are shown and error bars indicate SD. (<b>B</b>) LSECs were MCMV infected at indicated MOIs and ¼ of the SN was harvested for titration at 7, 11, 14 and 19 dpi, and substituted with medium ± IFNβ. Titration was performed on IFNAR^−/− MEFs. Histograms show average titers (PFU/mL) from replicates ± SD.</p

Inhibition of viral replication by IFNβ is reversible and occurs prior to immediate-early gene expression.

<p>LSECs were treated with IFNβ for 24 h and infected with MCMV^r. (<b>A</b>) Representative EYFP fluorescence microscopy of IFNβ treated (500 U/mL) or untreated LSECs at 7 dpi; MOI = 0.0001 is shown. (<b>B</b>) Twelve wells per condition were analyzed at multiple time points of infection. Wells containing as little as a single infected cell were classified as positive. (<b>C</b>) Cells were treated with 10, 100 or 500 U/mL of IFNβ for 24 h, infected at the indicated MOIs and the percentage of positive wells (of 12) was determined at 7 dpi. Histograms indicate average values from three independent experiments, error bars show SD. (<b>D</b>) IFNβ-treated LSECs were infected with an MOI of 0.0001. At dpi 7, IFNβ was removed in selected wells (+/− IFNβ) and wells were monitored for EYFP expression at the indicated time points post IFN retraction. The gain of positive wells as mean percentage from three independent experiments ± SEM is shown. (<b>E</b>) LSECs were pre-stimulated with 500 U/mL IFNβ and infected with 0.001 MOI of MCMV^r. SN were collected on 7, 11, 14 and 19 dpi and titrated on MEFs. Graphs show average titers (PFU/mL) from duplicates (± SD). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003962#ppat.1003962.s001" target="_blank">Figure S1</a>.</p

MCMV replication is completely blocked in IFNβ-responsive cells.

<p>NIH3T3 cells expressing the IRF7–mCherry fusion protein were treated with 500 U/mL IFNβ for 24 h and analyzed for mCherry expression by both fluorescence microscopy and flow cytometry. Representative microscopic pictures (<b>A</b>) and flow cytometry (<b>B</b>) of IFNβ-treated (white histogram) and untreated (grey histogram) cells are shown. (<b>C</b>) IRF7 reporter cells were cultured in the presence of IFNβ for 24 h and FACS sorted as responder (mCherry⁺) or non-responder (mCherry⁻) cells. Both cell populations were cultivated in absence (left), or presence of IFNβ (right), infected with MCMV Δm157 eGFP at 0.01 MOI. SN were collected at 0 (input virus), 2, 4 and 6 days post infection (dpi). Virus titrations were performed on IFNAR^−/− MEFs. Graphs show average titers (PFU/mL) of three independent experiments ± SEM.</p

Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency.

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNβ blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNβ is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNβ. Finally, IFNβ prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNβ is consistent with the establishment of CMV latency